I use BV2 cell line to record a calcium signal with a dye called Calbryte 520 AM. I added ATP into the perfusion system at 5 min, and I could see a peak in the figure. I did four times of this experiment, but the calcium baseline decreased continuously. Generally, the calcium baseline should be stable.
"Generally, the calcium baseline should be stable"...Why?
You are opening the shutter for more than 5 min. You can't expect the fluorescence signal to stay constant. From the little experience I have with Calcium imaging, your signal will have to decrease with time. I would limit the shutter opening time, say open it 10 to 15 sec before adding ATP. But Listen to what others who do Ca imaging more frequently might say :). Good luck!
Also, you might need to have a fast perfusion system for your rig.
Nice signal. The decrease is probably due to bleaching like both answers said. Continuous and more exponential than linear. Nothing dramatic you can either decrease the illumination power to reduce it or you can detrend it post hoc with your software or choice. The fact that it comes back up on the blue trace is a bit strange but probably due to the way you calculate your DeltaF/F0.
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